Pathogenic E. coli O-157 are 2 μm long and 1 μm wide rod-shaped bacteria, discovered in the United States in 1982.In Japan, an outbreak of O-157 infection occurred in 1996-1997,leading to a large number of deaths, and becoming a major social problem. This outbreak occurred because discovery of infection was delayed due to the average of seven days between oral infection via food, drinking water, patient excrement and such, the development of symptoms such as diarrhea, and the beginning of treatment. In addition, the toxicity of verotoxin produced by E. coli O-157 is several dozen times stronger than that of fugu blowfish. Another reason for this outbreak is that a rapid method for detecting verotoxins has not been fully explored.
Conventionally, known methods for detecting E. coli O-157 are based on three principles: antigen-antibody methods, PCR methods, and bioassay methods. Combinations of these methods have also been proposed, and several kits based on these principles are commercially available. In antigen-antibody methods, antigen-antibody reactions are performed on each protein of the six subunits that form one verotoxin, and thus measurements take a long time. In addition, since measurement errors may occur, accurate detection of verotoxins is difficult in certain cases. PCR methods determine the possible existence of verotoxins from E. coli gene fragments; verotoxins cannot be detected directly. Bioassay methods allow detection with a certain level of accuracy, however, are problematic in that the procedures are complicated and verotoxin analysis requires approximately three to four days.
Thus, many of the currently known methods for detecting verotoxins only investigate or suggest the existence of E. coli cells, requiring time-consuming and laborious analysis, yet yielding unreliable results.
On the other hand, a biosensor in which sugar chains are immobilized on the surface of a substrate such as gold has been proposed as a general biosensor that uses sugar compounds (U.S Pat. No. 6,231,733). This biosensor has a structure in which groups comprising sugar chains bind directly and covalently to a gold surface. In this sensor, low molecular weight compounds such as hydrocarbons are initially bound to sugar chains, thiol groups are bound to the sugar chain portions, and then the sugar chain portions are immobilized onto the substrate by direct covalent bonding or adsorption between the gold surface and the thiol group (SH).
Thiol group-mediated bonds are stable. However, sugar chain derivatives, in contrast to antigens, antibodies, enzymes, and such, comprise many hydroxyl groups, and also have complex three-dimensional structures. Thus introducing thiol groups to the sugar chain portions requires many steps and a great deal of effort. This is problematic in that thiol groups cannot be easily introduced to sugar chains. Alternatively, it is theoretically possible to first introduce groups comprising thiol groups to the gold surface, and then bind separately synthesized sugar chain portions to these. However, the introduction of sugar chain portions to the gold surface in this method is inefficient, and it hence cannot be practically employed.
To bind sugar chain portions comprising thiol groups to a gold surface, a large number of sugar compounds should be produced and immobilized onto a substrate. However, it is generally difficult to uniformly and simultaneously introduce a large amount of sugar chain portions to a gold surface, and thus pinholes may be produced, or alternatively the procedure of the binding reaction must be repeated.
On the other hand, the sugar compound detection reagents previously developed by the present inventors (Unexamined Published Japanese Patent Application (JP-A) No. 2001-342197) comprise sugar chain portions that can recognize and bind to verotoxins, and structures in which hydrocarbon chains are bound to these sugar chains as aglycone portions. The hydrocarbon chains of these aglycone portions are immobilized to a hydrophobically treated substrate by weak bonds, specifically hydrophobic bonds. Therefore, they have poor stability and durability, and the detection reagents can sometimes partially detach from the substrate when immersed for a long time in water or buffer, or when repeatedly used.
In particular, sensor chips to which toxins are bound normally have sugar chains bound via monolayer films. Therefore, when sensor chips are washed after a single use, the sugar compounds immobilized to the substrate surface (JP-A No. 2001-342197) can detach. Thus it is necessary to re-accumulate sugar compounds on to the gold substrate.
After producing monolayer films by this method, the films must be transferred to (or accumulated on) appropriate substrates. However, when transferring and immobilizing a large amount of sugar compound on to a substrate, it is difficult to carry out many treatments at once. Specifically, there is a limit to the process of accumulating a large amount of monolayer film on hydrophobically treated gold surfaces in a short period of time, and thus this process is far from practical.